The Molecular Mechanism Of Symbiont-mediated Anti-plant Defense Reaction In Wheat Aphid Sitobion Avenae

Sitobion avenue(Fabricius)is a dominate species in the wheat growing fields of China and causes numerous economic losses.However,the strong adaptability of aphid leads to lacking of resistance gene,making conventional insect-resistant breeding difficultly.From now on,the reason of aphid strong adaptability and how aphid overcome the host plant defense reaction still unrevealed.With the long-term co-evolutionary process,insects and their endosymbionts established a close mutualistic relationship.The resident endosymbionts could f)zlcilitate their host to regulate nutrition and improve the adaptation under the adverse environment.Plants initiate anti-herbivore defense responses when attacked by herbivores.Therefore,herbivorous insects have evolved strategies to circumvent plant defense responses.Insect symbionts as hidden players in insect-plant interactions and play an important role in the "arm race" between insect and host plant interaction.However,the potential function of symbiont in the aphid-host plant interaction still unclearly.Thus,our study used wheat aphid S.avenae,secondary symbiont Hamiltonella defensa and wheat as research objects,applying the technologies such as biology,molecular biology and enzyme detection to reveal the anti-plant responses strategics mediated by the H.defensa in wheat aphid S.avenae.Otherwise,we also performed the proteomic analysis to identify the different expressed or specific protein between Hamiltonella-infected and Hamiltonella-free aphid saliva,providing a theoretical basis for further in-depth study of symbiont-mediated interactions between aphid and wheat The results are as follows:1.Identification of symbiont diversity in different geographical clones of S.avenaeThe diversity of S-symbiont infections of S.avenae was investigated from 17 isofemale strains by specific PCR detection.In these isofemale clones,no single S-symbiont infected clone was detected.Interestingly,the infection of S-symbionts among the different wheat-producing provinces differed somewhat,but samples from different places in the same province showed a high degree of consistency2.Effect of H.defensa on fitness and relative abundance of Buchnera aphidicolaUsing artificial intection and antibiotic treatment,a zamiltonella-rcinfected clone(DZ-H)and Hamiltonella-curcd(DZ-HT)clone were established and compared to the Hamiltonclla-free clone(DZ)in terms of ecological fitness.The results showed that infection with H.defensa increased the fitness of S.avenae,which lead to increases in adult weight,percent ot wingless individuals and number of offspring.Furthermore,quantitative PCR showed that the density of B.aphidicola of the DZ-H clone was significantly higher than the DZ clone on server time periods.Meanwhile,the sharp decline time of B.aphidicola density occurred earlier in the DZ clone than the DZ-H clone.Additionally,the density dynamic of H.defensa in the DZ-H clone is also similar with B.aphidicola density dynamics,presenting earlier increase and later decreased trend.3.Anti-plant defense response strategies mediated by Hamiltonella defensaFluorescence in situ hybridization(FISH)revealed that H.defensa,which located on the periphery of bacteriocytes where the primary symbiont B.aphidicola was housed.Meanwhile,H.defensa also detected in the aphid hind-gut.Furthermore,gene expression studies by real time RT-PCR showed that feeding by the DZ?H clone suppressed the salicylic acid(SA)-and jasmonic acid(JA)-related defense pathways in wheat plants relative to those in the DZ and DZ-HT controls.Additionally,after feeding by the DZ-H clone,the enzyme activity levels of the defense-related enzymes polyphenol oxidase(PPO)and peroxidase(POD)in wheat plants were significantly decreased compared with the levels observed after feeding by the DZ-H and DZ-HT clones.However,the aphid detoxification enzyme activity levels were significantly increased after infecting with H.defensa.4.Plant-mediated horizontal transmission of H.defensa in the wheat aphid S.avenaeFluorescence in situ hybridization(FISH)revealed that H.defensa was observed in aphid stylets and plant phloem.H.defensa persisted in wheat leaves for at least 10 days.Most Homiltonella-uninfected aphids became infected with H.defensa after sustained feeding on infected plant leaves and showed almost 100%stable vertical transmission over the next five generations.These horizontal transmission experiments were replicated using two other plants,rice and corn,and two different wheat aphid species,Rhopalosiphum padi and Schizaphis graminum.5.Comparative proteomic analysis of Hamiltonella-infecied and Hamiltonello-free aphid salivaWe collected the watery saliva of Hamiltonella-infeceed and Hamiltnella-free S.avenae by feeding with 20%surcrosc solution and identified the major proteins by protcomic analysis.A total of 255 proteins were identified,which were divided into 8 function categories,including shealth protein,enzymes,transporter,nucleic acid binding protein,ribosomal protein and unknown protein.Among them,43.1%proteins are origin from bacteria symbionts.The diversity and number of proteins in Hamiltonella-iniected aphid saliva was higher than Hamiltonella-free,meanwhile,numerous oxidoreductase and protease were identified in Hamiltonella-infected aphid saliva.Noteworthy,46 secreted proteins with signal peptides were identified in 255 proteins and the 2 secreted proteins were origin from H.defensa.Therefore,we speculate that the reason for increased adaptation of aphid after infection with H.defensa may be related to the abundance and diverse specific proteins in saliva.The role of these specific proteins in the interaction between aphid and wheat needs further investigation.